Kenji Kai

Safranal, a novel protein tyrosine phosphatase 1B inhibitor, activates insulin signaling in C2C12 myotubes and improves glucose tolerance in diabetic KK-Ay mice.

SCOPE Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signaling by tyrosine dephosphorylation of insulin receptor, and its increased activity and expression is implicated in the pathogenesis of insulin resistance. Hence, PTP1B inhibition is anticipated to improve insulin resistance in type 2 diabetic subjects. The aim of this study was to find a novel PTP1B inhibitor from medicinal food and to evaluate its antidiabetic effects. METHODS AND RESULTS We found that saffron (Crocus sativus L.), which is used both as a spice and as a traditional medicine, potently inhibits PTP1B activity. Analyses of saffron extracts demonstrated that safranal, the saffrons aroma compound, is a principal PTP1B inhibitor, and induces a ligand-independent activation of insulin signaling in cultured myotubes. Our data implied that the molecular mechanism underlying the inactivation of PTP1B could be attributed to the covalent modification of the catalytic cysteinyl thiol by safranal through a Michael addition. Furthermore, safranal significantly enhanced glucose uptake through the translocation of glucose transporter 4. We also demonstrated that 2-wk oral administration of 20 mg/kg/day safranal improved impaired glucose tolerance in type 2 diabetic KK-A(y) mice. CONCLUSION Our results strongly suggest the usefulness of safranal in antidiabetic treatment for type 2 diabetic subjects.

Methyl 3-Hydroxymyristate, a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum.

Ralstonia solanacearum, a plant pathogenic bacterium causing “bacterial wilt” on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3‐hydroxypalmitate (3‐OH PAME) was previously identified as the QS signal in strain AW1. However, 3‐OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)‐methyl 3‐hydroxymyristate [(R)‐3‐OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)‐3‐OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types—(R)‐3‐OH MAME or (R)‐3‐OH PAME. These results demonstrate that (R)‐3‐OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.

Production of the Quorum-Sensing Molecules N-Acylhomoserine Lactones by Endobacteria Associated with Mortierella alpina A-178

Gram‐negative bacteria communicate with one another using N‐acylhomoserine lactones (AHLs) as signaling molecules. This mechanism, known as quorum sensing (QS), is needed to develop pathogenicity, as well as symbiotic interactions with eukaryotic hosts, such as animals and plants. Increasing evidence indicates that certain bacteria, namely endobacteria, also inhabit fungal cells and establish symbiotic relationships with their hosts. However, it has not been clear whether bacterial QS acts in developing the relationships. Here we describe the isolation and identification of N‐heptanoylhomoserine lactone and N‐octanoylhomoserine lactone from the culture broth of the zygomycete fungus Mortierella alpina A‐178. This suggested the presence of endobacteria in the fungus, as was confirmed by PCR, fluorescence in situ hybridization, and transmission electron microscopy. Two major bands obtained by PCR‐denaturing gradient gel electrophoresis showed sequence identity to genes in the β‐proteobacterium Castellaniella defragrans (100 %) and the Gram‐positive bacterium Cryobacterium sp. (99.8 %). The production of AHLs depended on the presence of endobacteria and was induced in response to the increase in the concentration of AHLs, suggesting that the bacterium conducts AHL‐mediated QS in the fungus. This paper is the first to report the production of AHLs by endofungal bacteria and raises the possibility that QS plays roles in the development of fungus–endobacterium symbiosis.

Inhibitory activity against urease of quercetin glycosides isolated from Allium cepa and Psidium guajava.

Methanolic extracts of edible plants and seaweeds were tested for their inhibitory activity against Jack bean urease. Quercetin-4′-O-β-D-glucopyranoside was isolated from Allium cepa as a urease inhibitor with an IC50 value of 190 μM-. Quercetin and two quercetin glycosides, avicularin and guaijaverin, were isolated from Psidium guajava as urease inhibitors with respective IC50 values of 80 μM-, 140 μM-, and 120 μM-.

β‐Caryophyllene attenuates palmitate‐induced lipid accumulation through AMPK signaling by activating CB2 receptor in human HepG2 hepatocytes

SCOPE Nonalcoholic fatty liver disease is currently the most common chronic liver disease worldwide, characterized by excessive hepatic lipid accumulation without significant ethanol consumption. We have performed a screening for medicinal foods that inhibit hepatocytic lipid accumulation through activation of AMP-activated protein kinase (AMPK), which is a critical regulator of the hepatic lipid metabolism. METHODS AND RESULTS We found that clove (Syzygium aromaticum), which is commonly used as a spice, markedly inhibits palmitate-inducible lipid accumulation in human HepG2 hepatocytes. Analyses of the clove extracts found that β-caryophyllene, an orally-active cannabinoid, is the principal suppressor of the lipid accumulation, and stimulates the phosphorylation of AMPK and acetyl-CoA carboxylase 1 (ACC1). Our data also showed that β-caryophyllene prevents the translocation of sterol regulatory element-binding protein-1c (SREBP-1c) into the nucleus and forkhead box protein O1 (FoxO1) into the cytoplasm through AMPK signaling, and consequently, induces a significant downregulation of fatty acid synthase (FAS) and upregulation of adipose triglyceride lipase, respectively. Moreover, we demonstrated that the β-caryophyllene-induced activation of AMPK could be mediated by the cannabinoid type 2 receptor-dependent Ca2+ signaling pathway. CONCLUSION Our results suggest that β-caryophyllene has the potential efficacy in preventing and ameliorating nonalcoholic fatty liver disease and its associated metabolic disorders.

New Chrodrimanin Congeners, Chrodrimanins D–H, from YO-2 of Talaromyces sp.

Four new meroterpenoids, named chrodrimanins D-G (4-7), and one known compound, renamed chrodrimanin H (8), were isolated from okara (the insoluble residue of whole soybean) that had been fermented with the YO-2 strain of Talaromyces sp. Their structures were elucidated by spectroscopic methods. Chrodrimanins D (4), E (5), and F (6) showed insecticidal activity against silkworms with respective LD(50) values of 20, 10, and 50 µg/g of diet.

Involvement of Ralfuranone Production in the Virulence of Ralstonia solanacearum OE1-1

Ralstonia solanacearum causes a destructive disease called “bacterial wilt” in numerous plant species. Its virulence is controlled by the transcriptional regulator PhcA, the activity of which is, in turn, regulated in a cell‐density dependent manner, termed quorum sensing. We herein described the identification and characterization of ralfuranones J–L, new PhcA‐regulated secondary metabolites, and the known derivatives, ralfuranones A and B, from R. solanacearum strain OE1‐1. Their structures were determined by spectroscopic and chemical methods. These ralfuranones were also detected in vascular exudates from host plants infected with OE1‐1. Deletion of ralA, which encodes an enzyme for ralfuranone biosynthesis, reduced the virulence of OE1‐1 in tomato plants. Virulence was restored by complementation of the ralA gene. The results suggest that ralfuranones play important roles in the virulence of OE1‐1.

Determination of Absolute Structures of Cyclic Peptides, PF1171A and PF1171C, from Unidentified Ascomycete OK-128

Two cyclic peptides, PF1171A (1) and PF1171C (2), were isolated from okara that had been fermented with unidentified ascomycete OK-128. Their absolute configurations were determined by Marfey’s method. These peptides showed paralytic activity against silkworms.

GluCl a target of indole alkaloid okaramines: a 25 year enigma solved

In 1989, indole alkaloid okaramines isolated from the fermentation products of Penicillium simplicissimum were shown to be insecticidal, yet the mechanism of their toxicity to insects remains unknown. We therefore examined the action of okaramine B on silkworm larval neurons using patch-clamp electrophysiology. Okaramine B induced inward currents which reversed close to the chloride equilibrium potential and were blocked by fipronil. Thus it was tested on the silkworm RDL (resistant-to-dieldrin) γ-aminobutyric-acid-gated chloride channel (GABACl) and a silkworm L-glutamate-gated chloride channel (GluCl) expressed in Xenopus laevis oocytes. Okaramine B activated GluCl, but not RDL. GluCl activation by okaramines correlated with their insecticidal activity, offering a solution to a long-standing enigma concerning their insecticidal actions. Also, unlike ivermectin, okaramine B was inactive at 10 μM on human α1β2γ2 GABACl and α1β glycine-gated chloride channels and provides a new lead for the development of safe insect control chemicals.

A New Meroterpenoid, Chrodrimanin C, from YO-2 of Talaromyces sp.

The new meroterpenoid, chrodrimanin C (3), together with chrodrimanins A (2) and B (1) were isolated from okara (the insoluble residue of whole soybean) that had been fermented with strain YO-2 of Talaromyces sp. Their structures were elucidated by spectroscopic methods. The partial structures of 1 essential for exhibiting insecticidal activity were investigated by using a silkworm assay. The absolute configuration of 1 was also determined.